Two-part device for T-cell receptor synthesis and stable genomic integration to TCR-presenting cells

Abstract

The present invention relates to a two-part device, wherein a first part is a multicomponent TCR ORF reconstitution and engineering system (TORES), and a second part is a multicomponent engineered TCR-presenting cell system (eTPCS).

The invention claimed is: 
     
       1. A two-part device, wherein a first part is a multicomponent T cell receptor (TCR) open reading frame (ORF) reconstitution and engineering system (TORES), and a second part is a multicomponent engineered TCR-presenting cell system (eTPCS), wherein:
 (I) the TORES comprises three separate components:
 (a) a vector carrying variable and constant (V-C) T-cell receptor (TCR) gene segments, designated as V-C entry vector component 1A; 
 (b) a vector carrying joining (J) TCR gene segments, designated as J donor vector component 1B; and 
 (c) a complementarity-determining region 3 (CDR3) vector designated as component 1C, comprising an oligonucleotide duplex encoding CDR3 (odeCDR3), 
 
 wherein, when recombined, components 1A, 1B, and 1C provide a genetic integration vector, designated as genetic integration vector component 2C or 2E, each encoding an analyte TCR ORF of alpha, beta, delta, or gamma selected from:
 (a) a native TCR chain; 
 (b) a sequence-diversified TCR chain; and 
 (c) a synthetic TCR chain; 
 
 (II) the eTPCS comprises three separate components:
 (a) an eTPC component designated as eTPC component 2A, wherein eTPC component 2A lacks endogenous expression of TCR chains alpha, beta, delta and gamma, and
 expresses cluster of differentiation 3 (CD3) proteins which are conditionally presented on the surface of the cell only when the cell expresses a complementary pair of TCR chains, and 
 
 (b) a genomic receiver sites component, designated as components 2B and 2D, each for integration of a single ORF encoding one analyte TCR chain of alpha, beta, delta or gamma, wherein the genomic receiver site components 2B and 2D are each selected from:
 (a) a synthetic construct designed for recombinase mediated cassette exchange (RMCE); and 
 (b) a synthetic construct designed for site directed homologous recombination; and 
 (c) genetic integration vector component 2C or 2E; and 
 
 
 wherein the genetic integration vector components 2C and 2E each recombine with the genomic receiver site components 2B and 2D, respectively. 
 
     
     
       2. The two-part device of  claim 1 , wherein the eTPCS provides one or more analyte eTPC in which one or more TORES-derived analyte TCR chains are presented, and the one or more analyte eTPC is selected from
 (a) an eTPC expressing a TCR pair (eTPC-t); 
 (b) an eTPC expressing one TCR chain (eTPC-x); and 
 (c) one or more libraries of (a) or (b). 
 
     
     
       3. The two-part device of  claim 2 , wherein a pair of analyte TCR chains are expressed as TCR surface proteins (TCRsp) in complex with CD3 by an analyte eTPC. 
     
     
       4. The two-part device of  claim 1 , wherein the V-C entry vector component 1A comprises:
 (a) an origin of replication, 
 (b) a first positive selection marker, 
 (c) one or more 5′ genetic elements, 
 (d) a Kozak Sequence, 
 (e) a TCR variable gene segment, 
 (f) a first Type IIS sequence, for site specific recognition and cleavage by a Type IIS restriction enzyme, 
 (g) a negative selection marker, 
 (h) a second Type IIS sequence, 
 (i) a TCR constant gene segment, and 
 (j) one or more 3′ genetic elements. 
 
     
     
       5. The two-part device of  claim 4 , wherein the J donor vector component 1B comprises:
 (a) an origin of replication, 
 (b) a second positive selection marker, 
 (c) a third Type IIS sequence, 
 (d) a TCR Joining gene segment, 
 (e) a C part, corresponding to a small 5′ portion of a constant gene segment, and 
 (f) a fourth Type IIS sequence. 
 
     
     
       6. The two-part device of  claim 5 , wherein the first positive selection marker of component 1A and the second positive selection marker of component 1B are different and are selected from an antibiotic resistance gene and an auxotroph complementing gene. 
     
     
       7. The two-part device  claim 5 , wherein the CDR3 vector component 1C comprises:
 (a) a first single-stranded overhang sequence complementary to the first Type IIS restriction enzyme recognition and cleavage site in the V-C entry vector component 1A, 
 (b) a double-stranded segment encoding a TCR CDR3 region and devoid of the negative selection element present in V-C entry vector component 1A, and devoid of the Type IIS restriction sequences present in V-C entry vector component 1A and J donor vector component 1B, and 
 (c) a second single-stranded overhang sequence complementary to the third Type IIS restriction enzyme recognition and cleavage site in the J donor vector component 1B; 
 or the CDR3 vector component 1C comprises 
 (d) a single double-stranded DNA molecule encoding a TCR CDR3 flanked by Type IIS restriction enzyme sites such that when cleaved generates a product comprising V-C, CDR3, and J (V-CDR3-J-C TCR ORF). 
 
     
     
       8. The two-part device of  claim 4 , wherein the 5′ genetic element of the V-C entry vector component 1A further comprises one or more elements selected from:
 (a) a gene cis/acting element, 
 (b) a heterospecific recognition site for recombinase enzymes, 
 (c) a 5′ homologous recombination arm for a genomic site of interest, 
 (d) a mRNA splice acceptor site, 
 (e) an internal ribosomal entry site, and 
 (f) an epigenetic insulator sequence, 
 
       wherein the 5′ genetic element must contain at least element (b) or element (c). 
     
     
       9. The two-part device of  claim 8 , wherein the negative selection marker in the V-C entry vector component 1A is selected from one or more of the following:
 (a) a restriction enzyme recognition site not contained elsewhere in the first component or within the TCR joining gene segment, 
 (b) a gene encoding a conditional bactericidal agent, and 
 (c) a reporter element. 
 
     
     
       10. The two-part device of  claim 4 , wherein the 3′ genetic element of the V-C entry vector component 1A further comprises one or more elements selected from:
 (a) a terminator element, 
 (b) a heterospecific recognition site for recombinase enzymes, 
 (c) a 3′ homologous recombination arm for a genomic site of interest, 
 (d) a mRNA splice donor site, 
 (e) an internal ribosomal entry site, and 
 (f) an epigenetic insulator sequence 
 wherein the 3′ genetic element must contain at least element (b) or element (c). 
 
     
     
       11. The two-part device of  claim 1 , wherein the eTPC component 2A is a cell that lacks endogenous surface expression of at least one family of analyte antigen-presenting complexes (aAPX) or analyte antigenic molecule (aAM). 
     
     
       12. The two-part device of  claim 11 , wherein the family of aAPX is selected from any of the following:
 (a) human leukocyte antigen (HLA) class I; 
 (b) HLA class II; and 
 (c) non-HLA antigen-presenting complex. 
 
     
     
       13. The two-part device of  claim 12 , wherein the genomic receiver site components 2B and 2D each comprise at least one of the following genetic elements:
 (a) heterospecific recombinase sites; 
 (b) homologous arms; 
 (c) a eukaryotic promoter; 
 (d) a eukaryotic conditional regulatory element; 
 (e) a eukaryotic terminator; 
 (f) a selection marker; 
 (g) a splice acceptor site; 
 (h) a splice donor site; 
 (i) a non-protein coding gene; 
 (j) an insulator; 
 (k) a mobile genetic element; 
 (l) a meganuclease recognition site; 
 (m) an internal ribosome entry site (IRES); 
 (n) a viral self-cleaving peptide element; and 
 (o) a Kozak consensus sequence. 
 
     
     
       14. The two-part device of  claim 12 , wherein the genomic receiver site components 2B and 2D are for RMCE integration of a single ORF and each comprises:
 (a) a eukaryotic promoter; 
 (b) a pair of heterospecific recombinase sites that recombine with those of the genetic integration vector component 2C or 2E; 
 (c) a Kozak consensus sequence; 
 (d) a selection marker; and 
 (e) a eukaryotic terminator. 
 
     
     
       15. The two-part device of  claim 12 , wherein the two-part device comprises the genetic integration vector components 2C and 2E that are each for RMCE integration of a single ORF and each comprises the following genetic elements contributed by the V-C entry vector component 1A:
 (a) a pair of heterospecific recombinase sites that recombine with those of the genomic receiver site components 2B or 2D; 
 (b) a Kozak consensus sequence; and 
 (c) a TCR ORF reconstituted by operation of the TORES. 
 
     
     
       16. The two-part device of  claim 12 , wherein the two-part device comprises the genetic integration vector components 2C and 2E and the TORES provides a single analyte TCR chain pair. 
     
     
       17. The two-part device of  claim 16 , wherein the analyte TCR chain pair encoding sequences are from:
 (a) paired sequencing of TCR chain ORF sequence(s) from primary T-cells and reconstitution in the part 1 TORES; 
 (b) unpaired sequencing of TCR chain ORF sequence(s) from primary T-cells and reconstitution in part 1 TORES; or 
 (c) synthetic TCR chain ORF sequence(s) generated by operation of the TORES. 
 
     
     
       18. The two-part device of  claim 16 , wherein the genetic integration vector components 2C and 2E are combined with the eTPC component 2A to integrate two complementary analyte TCR chains encoded in the genetic integration vector components 2C and 2E into the genomic receiver site components 2B and 2D, thereby obtaining a cell that expresses an analyte TCRsp on its surface. 
     
     
       19. The two-part device of  claim 16 , wherein the genetic integration vector component 2C is combined with the eTPC component 2A to integrate one analyte TCR chain encoded in the genetic integration vector component 2C into the genomic receiver site component 2B, thereby obtaining a cell, designated eTPC-x, expresses a single TCR chain. 
     
     
       20. The two-part device of  claim 19 , wherein the genetic integration vector component 2E is combined with the eTPC-x to integrate one analyte TCR chain encoded in the genetic integration vector component 2E that is complementary to the TCR chain expressed in the eTPC-x into the genomic receiver site component 2D of the eTPC-x, thereby obtaining a cell that expresses a TCRsp on its surface. 
     
     
       21. The two-part device of  claim 12 , wherein the two-part device comprises the genetic integration vector components 2C and 2E and the TORES provides a library of analyte TCR chain pairs encoded by the genetic integration vector components 2C and 2E. 
     
     
       22. The two-part device of  claim 11 , wherein the aAM is selected from:
 (a) a polypeptide or complex of polypeptides translated from the analyte antigenic molecule ORF(s); 
 (b) a peptide from a polypeptide translated from the analyte antigenic molecule ORF(s); 
 (c) a peptide from the component A proteome; 
 (d) a polypeptide from the component A proteome; or 
 (e) a metabolite from the component A metabolome. 
 
     
     
       23. The two-part device of  claim 11 , wherein the eTPC component 2A expresses cluster of differentiation 4 (CD4) or cluster of differentiation 8 (CD8). 
     
     
       24. The two-part device of  claim 11 , wherein the eTPC component 2A expresses TCR co-receptors. 
     
     
       25. The two-part device of  claim 11 , wherein the eTPC component 2A expresses cluster of differentiation 28 (CD28) or cluster of differentiation 45 (CD45). 
     
     
       26. The two-part device of  claim 11 , wherein the two-part device comprises the genetic integration vector components 2C and 2E wherein each comprises at least one of the following genetic elements:
 (a) heterospecific recombinase sites; 
 (b) homologous arms; 
 (c) a eukaryotic promoter; 
 (d) a eukaryotic conditional regulatory element; 
 (e) a eukaryotic terminator; 
 (f) a selection marker; 
 (g) a selection marker of integration; 
 (h) a splice acceptor site; 
 (i) a splice donor site; 
 (j) a non-protein coding gene; 
 (k) an insulator; 
 (l) a mobile genetic element; 
 (m) a meganuclease recognition site; 
 (n) an internal ribosome entry site (IRES); 
 (o) a viral self-cleaving peptide element; 
 (p) an antibiotic resistance cassette; 
 (q) a bacterial origin of replication; 
 (r) a yeast origin of replication; 
 (s) a cloning site; and 
 (t) a Kozak consensus sequence. 
 
     
     
       27. The two-part device of  claim 1 , wherein: the eTPC component 2A:
 (a) lacks endogenous expression of TCR chains alpha, beta, delta and gamma, 
 (b) expresses CD3 proteins which are conditionally presented on the surface of the cell only when the cell expresses a complementary pair of TCR chains, and 
 (c) contains the genomic receiver site component 2B for integration of a single ORF encoding at least one analyte TCR chain of alpha, beta, delta or gamma, or two ORFs encoding pair of analyte TCR chains, 
 and the genetic integration vector component 2C recombines with the genomic receiver site component 2B, and 
 wherein the genetic integration vector component 2C encodes:
 (i) a single ORF encoding at least one analyte TCR chain of alpha, beta, delta and/or gamma; or 
 (ii) two ORFs encoding a pair of analyte TCR chains. 
 
 
     
     
       28. The two-part device of  claim 27 , wherein the single ORF (i) or two ORFs (ii) of the genetic integration vector component 2C encodes a selection marker of integration, such that the analyte TCR chains can be expressed as a TCR surface protein (TCRsp) in complex with the CD3 on the surface of the eTPC component 2A. 
     
     
       29. The two-part device of  claim 1 , wherein the eTPC component 2A further contains a synthetic genomic TCR-stimulation response element, designated as component 2F and selected from:
 (a) a single component synthetic construct containing at least one native promoter or at least one synthetic promoter and at least one reporter; and 
 (b) a multi-component synthetic construct designed with at least one native promoter or at least one synthetic promoter and at least one reporter, 
 and wherein activation of (a) or (b) is dependent on at least one signal transduction pathway selected from a synthetic pathway, a native pathway or a combination thereof. 
 
     
     
       30. The two-part device of  claim 1 , wherein the genomic receiver site components 2B and 2D are synthetic constructs designed for recombinase mediated cassette exchange (RMCE). 
     
     
       31. The two-part device of  claim 1 , wherein the two-part device generates at least one cell, designated an analyte eTPC-t, that expresses a TCRsp on its surface. 
     
     
       32. The two-part device of  claim 1 , wherein genetic integration vector components 2C and 2E each encodes a selection marker of integration, such that the analyte TCR chains can be expressed as TCR surface protein (TCRsp) in complex with the CD3 on the surface of the eTPC component 2A.